46,XY女性性反转患者SRY基因启动子区一个新的点突变及其功能分析

通过ＤＮＡ序列测定在一名４６,ＸＹ女性性反转患者ＳＲＹ基因启动子区发现了一个新的突变：ｎｔ．－８１Ｇ→Ａ．该突变不见于正常男性,因此不是ＤＮＡ多态性．为了检测这一点突变对ＳＲＹ基因表达功能的影响,构建了分别由正常或突变的人ＳＲＹ基因启动子区片段调控氯霉素乙酰转移酶（ＣＡＴ）报告基因表达的两个质粒,寡核苷酸探针杂交证实该启动子片段正常或携带有Ｇ→Ａ突变．这两个质粒分别与ｐＳＶ－β－半乳糖苷酶内对照质粒共转染ＨｅＬａ细胞后,瞬间表达分析显示这一突变对ＣＡＴ酶活性水平无显著影响（０．５０＞Ｐ＞０．２０）．上述正常和突变的ＳＲＹ基因启动子片段与Ｋ５６２细胞核抽提物的凝胶阻滞实验也表明,突变对Ｋ５６２细胞核蛋白与ＳＲＹ基因启动子区的结合影响不大．研究ＳＲＹ基因的表达调控对阐明人的性别决定机制及性反转的病理机制具有重要意义     

No evidence of mutations in four candidate genes for male sex determination/differentiation in sex-reversed XY females with campomelic dysplasia.

Campomelic dysplasia (Cd) occurs combined with sex reversal resulting in XY females. The recent identification of candidate genes for sex determination/differentiation and of a sex determining region on the human Y chromosome prompted the authors to study these genes for mutations in patients with Cd and sex reversal. In a total of five cases, no evidence for a mutation in the genes SRY, ZFY, ZFX, MEA and some anonymous Y-linked sequences was found. In addition to Southern analysis, gene expression of ZFY, ZFX and MEA was found to be normal as well. It is concluded that sex reversal in this condition is due to mutation in a so far unidentified gene which may act secondary to the testis-determining factor (TDF).     

Genetics of mammalian sex determination: some unloved exceptions

The genetics of sex determination is a child of the twentieth century, which overturned the previously held view that sex was determined by the environment. The last quarter of the century witnessed an active search for sex-determining genes in mammals. Although successful, the modus operandi of these genes remained unknown, and the relationship between the sex-determining systems of mammals and other vertebrates remained enigmatic. To overcome these problems, scientists in the 21st century should heed William Bateson's counsel to treasure exceptions, for they point the way to progress. One exception to conventional concepts of sex determination is the bilaterally asymmetrical distribution of ovaries and testes in true hermaphroditism. Ovaries favour the left side in humans and the right side in mice. Observations suggesting that a reversal of asymmetry may occur with increasing organ size may point to a possible explanation. A reevaluation is also required regarding the beginning of sex differentiation, in view of mounting evidence of a sex difference in growth rates of early embryos. Another question to be settled is whether the function of SRY is confined to the fetal gonad. The recent demonstration that Sry induces cell proliferation in the fetal mouse gonad (Schmahl et al., 2000) further emphasizes the importance of differential growth in sex determination and differentiation. It is suggested that SRY represents an additional growth-promoting gene sequestered by mammals to enable the XY embryo to undergo male sex differentiation in the female hormonal environment of the uterus. An increased awareness of the relationship between growth and gonadal differentiation should lead to a better understanding of sex determination in mammals and an ability to relate the function of sex-determining genes to the effects of environmental factors. J. Exp. Zool. 290:484-489, 2001.     

Mammalian sex reversal and intersexuality: deciphering the sex-determination cascade

The sex-determination cascade constitutes a model of the exquisite mechanisms of gene regulation that lead to the development of mammalian embryos. The discovery of the sex-determining region of the Y chromosome (SRY) in the early 1990s was the first crucial step towards a general understanding of sex determination. Since then, several genes that encode proteins with a role in this cascade, such as WT1, SF-1, SOX9, DAX-1 and WNT4, have been identified. Many of the interactions between these proteins have still to be elucidated, while, no-doubt, others are still to be identified. The study of mammalian intersexes forms a promising way towards the identification of the still-missing genes and a comprehensive view of mammalian sex determination. Intersexuality in the goat, studied for over a century, will, presumably, bring to light new genes involved in the female sex-determination pathway.     

Identifying genes for male sex determination in humans.

The convergence of genetic and molecular technologies has led to the identification of a number of genes for male sex determination. The observation of chromosomal translocations, deletions, and duplications in sex reversed individuals was instrumental for the positional cloning of SRY, SOX9, WT1, and DAX1. Cloning by protein-DNA interaction was required for the identification of SF1. The observation of an extended phenotype for the alpha thalassemia-mental retardation syndrome assigned a role for XH2 in the testicular determining process. Over the next several years, new sex determining genes will be identified by linkage analysis in large families with multiple sex reversed members, comparative genomic hybridization of sex reversed individuals, and database searches for genes that encode interacting proteins or paralogs of other species. Given the apparent differences in the sex determining mechanisms of even closely related species, the roles of all of these genes will require confirmation by demonstrating expression in human gonadal ridge at the critical time, and that mutations result in sex reversal.     

哺乳动物性别决定和性反转

目前已知SRY仅是涉及性别决定过程的基因之一.近年来又发现和克隆了许多可能参与性腺分化与发育的基因,如副中肾抑制基因MIS,也称抗副中肾激素基因AMH;SRY相关基因SOX9;编码甾类因子的基因SFI;X-连锁的DAX基因;Wilm′s肿瘤抑制基因WTI;以及X-连锁的剂量敏感基因DSS等,并新建立了性别决定的Z-基因模型,DSS-基因模型和Jimenez等的模型,较合理地解释了哺乳动物性别决定的分子机理和以前难以解释的各种奇特的性反转现象,使性别决定的研究取得了长足的进展,但仍有一些悬而未决的问题有待于进一步探索.     

Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.     

46,XY女性性反转患者SRY基因的两个新突变类型

Y染色体上的性别决定区域——SRY基因作为睾丸决定因子,可以调控男性性别发育过程。SRY基因是一种转录因子,属于带有高迁移率族蛋白家族,该家族成员包含能与DNA结合的HMG盒基序。已知SRY基因的缺失和点突变是造成XY女性性反转的病因之一。通过筛查10位中国46,XY女性性反转病人SRY基因的开放阅读框区域,探寻新的突变类型。用标准方法从外周血中抽提gDNA,通过聚合酶链式反应扩增SRY基因中部的609bp的DNA片段。扩增后的PCR片段被克隆到pUCm-T载体中,在ABI377-3自动测序仪上完成测序。运用限制性内切酶酶切分析的方法验证DNA测序的结果。结果表明,在两个患者的SRY基因中分别发现了新的核苷酸点突变,并都导致氨基酸替代。一个突变发生在SRY基因的5’端HMG盒外的核苷酸第113位腺嘌呤(A)被鸟嘌呤(G)取代,并导致谷氨酸被甘氨酸替换;另一个突变是第387位核苷酸发生T被A替换,该突变引起第129位的酪氨酸变成终止密码,她父亲的SRY序列被证明是正常的野生型。通过查询文献和人类基因突变数据库(HGMD),这两个突变都是以前未见报道过的新型SRY基因突变,并使因核苷酸替换引起SRY基因突变总数增加到45。     

HUMAN CHROMOSOME Y AND SRY

The precise location of the SRY gene on the human Y chromosome has been revealed through studies of sex reversal cases involving deletion, cross-linking and mutations of the SRY gene. Its DNA sequence and mechanism of action are being understood. Similarity of SRY with Sry of mice and its interaction with other genes in male sex determination are discussed.     

Molecular aspects of sex differentiation

Mammalian sex differentiation involves the action of a cascade of genes. Discovery of the sex-determining region of the Y chromosome (SRY) marked the beginning of the delineation of the genes in the cascade. Studies of the genetics of mammalian sex reversal and the embryogenesis of the mice are essential in this endeavor. A number of genes involved in the pathway have been identified and all except one of these genes have a putative role in male sex differentiation. Besides SRY being the master switch in male sex differentiation the hierarchical relationship of the genes identified are far from being understood. Similarly, our knowledge of the genetic regulation of female sex differentiation is minimal. Differential screening and gene expression profiling bring a new dimension to the pursuit with the identification of a number of genes previously unknown to be involved in sex differentiation. Wider application of functional genomic techniques and introduction of proteomic analyses are expected to shed light to our understanding of this complicated developmental process.     

Amplification and application of the HMG box of bovine SRY gene for sex determination

A fast and reliable method for bovine sexing has been developed through amplification of the bovine high motility group (HMG) box of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed according to the conserved bovine SRY HMG box sequence motif. In agarose gel electrophoresis, a normal bull showed 1 SRY band, and a normal cow showed no SRY band. After optimization, the PCR procedure for sex determination was applied to 14 embryo biopsies. The biopsied embryos were transferred into 14 recipient cows on the same day (day 7 of the estrus cycle) that the embryos were collected and sex of the calf was confirmed after parturition. Nine calves were born and anatomical sex corresponded to those sex determined by PCR in all cases (100% accuracy). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring and also allows a quick diagnosis for the XY-bovine offspring by amplification of the HMG box of the bovine SRY gene.     

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.     

Exclusion of candidate genes for canine SRY-negative XX sex reversal

In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.     

棕色田鼠性别决定研究进展

哺乳动物性别决定方式属于雄性异配型性别决定, 依赖于Y染色体, SRY基因是性别决定中最重要的基因。文章报道了棕色田鼠指名亚种有Y染色体, 但是Y染色体上没有SRY基因, 性别决定不依赖于SRY基因, 排除了R-spondin 1基因是性别决定基因, 同时讨论了棕色田鼠指名亚种SRY基因缺失后可能的性别决定 机制。     

Sex Determination in Reptiles: An Update

Sex determination and sex differentiation are two separate butrelated phenomena. Sex differentiation is a programmed cascadeof events in which the indifferent gonad develops as a testisor an ovary with the appropriate urogenital and secondary sexcharacters. Sex determination is the event that sets this cascadein motion. In placental mammals, there is good evidence thatsex is determined by a gene on the Y chromosome (SRY) that initiatestestis formation. In the absence of SRY an ovary develops. Thereare, however, examples of placental mammal that develop as normalmales with no detectable SRY. In reptiles, sex differentiationappears to be similar to mammals (i.e., the same genes and hormonesact ina similar manner), but sex determination is clearly verydifferent. Ovarian differentiation in placental mammals canoccur in the absence of estrogen or an estrogen receptor. Ovariandifferentiation in reptiles requires the presence of estrogen.In the absence of estrogen a testis develops. In TSD reptiles,embryos will develop as females when treated with estrogen evenif eggs are incubated at male-inducing temperatures, and conversely,will develop as males when estrogen synthesis is blocked ineggs incubated at female-inducing temperatures. A number ofother genes have also been shown to be important in mammaliansex determination. One of these genes, Sox9, which is expressedin differentiating mouse testis, has recently been found tobe expressed in embryonic reptile testis. Other genes that appearto be common to both mammals and reptiles in the sex determiningcascade are SF- 1, MIH, and possibly DAX-1. Current researchis now focused on how the gene that produces the enzyme necessaryfor estrogen synthesis (aromatase) is regulated in the embryosof reptiles with genetic or environmental sex determination.Controversial issues in reptilian sex determination are 1) therole of the brain in gonadal sex determination, and 2) the roleof steroid hormones in the yolk prior to sex determination